chapter 6-methods commonly used in medical genetics
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MethodsMethodscommonly used commonly used
in Molecular Geneticsin Molecular Genetics
outlineoutline
Nucleic acid extraction
Gel electrophoresis
Nucleic acid hybridization
Polymerase chain reaction
DNA recombination technology
Extraction of nucleic acid
DNA Extraction
RNA Extraction
Resource
steps
Resource
steps
DNA extractionDNA extraction All nucleated cells Fetal tissue: amniocentesis
chorionic villi samplingblood from umbilical cord
Adult tissue: Peripheral blood (white blood cells)cheek swab (buccal cells)skin cells, hair roots, tissue biopsysurgical specimens
Cell lysis
Removal of proteins and other components
Separation of nucleic acid
Washing and Dissolving the nucleic acid
Disruption of cells by chemicals or enzymes: detergents , proteinase K,……
Organic reagents (phenol, chloroform)Inorganic reagents (concentrated salt)
Ethanol or isopropanol
70% ethanol; sterile water
RNA extractionRNA extraction All nucleated cells Special tissue/cells at special development stage fresh tissue/cells
Brain, kidney, muscle, heart,
bone, liver, spleen, peripheral blood……
Cell lysis
Removal of proteins and other components
Separation of nucleic acid
Washing the nucleic acid
Inactivation of RNase to prevent RNA from degradation
Gel electrophoresisGel electrophoresis
Gel electrophoresis is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field.
electromotive force moves charged moleculesthrough a porous gel
separates molecules from each other on the basis of size, electric charge , other physical properties
separation depends on how the sample and gel are prepared
Gel
Agarose gela reversible matrix cross-linked by hydrogen bonds
Acrylamide gela permanent matrix cross-linked with methylene bridges
Translucent, porous, colloid, solid materials
composed of polymers of sugars
poured into slabs and columns and drawn into capillaries
Very stable
Pore size can also be controlled for, altering the migration
properties of the gel
Vertical Gel ElectrophoresisHorizontal Gel Electrophoresis
agarose gel electrophoresis
Monitoring the progress of the electrophoresis
xylene cyanol
migrates with ~5.0 kb fragments
bromphenol blue
migrates with fragments of a few hundred base pairs
Locating the DNA fragments in the gel
• wedges itself into the grooves of DNA and stays there•More base pairs mean more grooves, which in turn means moreEtBr can insert itself
•a fluorescent dye used for staining nucleic acids which has theproperty of fluorescing under UV light
Ethidium Bromide (EtBr or EB)
Polyacrylamide gel electrophoresisPAGE
Polyacrylamide has a smaller pore size than agarose, so it canResolve short ssDNA of the same length that differ in sequence.Resolve short fragments of dsDNA that differ in length by only a few oligonucleotides. Resolve fragments of denatured ssDNA that differ in length by only a single nucleotide.Resolve proteins.
Nucleic acid hybridizationNucleic acid hybridization
Definition Membrane hybridization
Southern blottingNorthern blottingDot/slot blottingIn situ hybridization
the bonding of two complementary single-stranded nucleic acid molecules according to base-pairing rules.
DNA / DNADNA / RNARNA / RNA
Probe used to detect probe’s complementary nucleic acid
To be detected nucleic acid
ProbeDefinition
Types of probe
Labeling
:a labeled DNA or RNA sequence used to detect the presence of a complementary sequence by molecular hybridization.
Radiative: Radioisotopes 32P, 33P, 35S
Nonradiative:Alkaline phosphatase;Biotin;
digoxigenin
DNA probe
RNA probe
Oligonucleotide probe
Type: DNA
Origin: Cell-based DNA cloning or PCR
Characteristics: double-stranded, 0.1~X00kb
Labeling: DNA polymerase-based DNA strand synthesis
DNA ProbesDNA Probes
RNA probesRNA probes
Type : RNA
Origin: Transcription from insert DNA cloned in
suitable vectors
Characteristics: single-stranded, x00bp
Labeling: RNA polymerase-based RNA synthesis
OligonucleotidesOligonucleotides probeprobe
Type: oligonucleotide
Origin: chemical synthesis
Characteristics: single-stranded, 15-50nt long
Labeling: end-labeling by polynucleotide kinase
Membrane hybridizationMembrane hybridization One nucleic acid component is affixed to
membrane; the other is in solution probe(s) affixed; sample in solution samples affixed; probe(s) in solution
Membrane material binds DNA or RNA nylon charged nylon nitrocellulose
Southern BlottingSouthern Blotting First described by the British biochemist E.
D. Southern DNA/DNA (detected DNA in gel) Steps applicationsDNA size change
DNA dosage change
Steps:Steps:
DNA extraction
REdigestion
Agarose gel electrophoresis
Transfer of DNA to membrane
Probe preparation
hybridization
Wash/rinse membrane
visualization
denaturation
Paper TowelsNylon Membrane
Buffer Buffer
GelWick
Transfer DNA from gel to DNA-binding membrane
Restriction EnzymesRestriction Enzymesoror
Restriction Restriction EndonucleaseEndonuclease (RE)(RE) Derived from bacteria, not human cells. Natural role - protect the bacteria containing them
against intruding DNA from other organisms (e.g. viruses).
Recognize a specific sequence of DNA and cut the DNA molecule within the recognition site or at some nearby site.type I, II, III
Allow the production of recombinant molecules
Restriction Restriction Endonuclease,typeEndonuclease,type IIII Recognizes specific, usually palindromic, DNA
sequence and cuts within the recognition site 4bp~8bp in length of the recognition sequence
KpnI
Eco RI
Ends created by RE digestionEnds created by RE digestion
Restriction Enzyme
Recognition Site
Ends generated
Type of End
Eco RI 5’GAATTC3’3’CTTAAG5’
5’G AATTC3’3’CTTAA G5’
5’ Overhang
Sma I 5’CCCGGG3’3’GGGCCC5’
5’CCC GGG3’3’GGG CCC5’
Blunt end
Sac I 5’GAGCTC3’3’CTCGAG5’
5’GAGCT C3’3’C TCGAG5’
3’ Overhang
Bam HI 5’GGATCC3’3’CCTAGG5’
5’G GATCC3’3’CCTAG G5’
5’ Overhang
DNA stained with ethidium bromide
autoradiograph of hybridized probe
Northern blotting
detect mRNA
electrophoresed in denatured agarose gel
Prevention from RNase contamination
applicationsDetect RNA expression
Determine RNA size
Quantify RNA expression
Dot/slot blotting
omit gel, spot known quantity of DNA or RNA on filter, probe with DNA, RNA or oligo useful for population screening: more samples on one
filter and less work than Southern blot (Yes or No)
Allele Specific Oligo (ASO) is known dot blottingDNA chip or DNA microarray
Two probes:20nt, single-stranded, synthetic oligonucleotide
Normal probe match precisely the normal DNA
Mutant probe match precisely the mutant DNA
ASO: Detect single base-pair change
A Autosomal disease
In situ hybridizationProbe, labeled by radioactivity or florescence, hybridizes to a chromosome spread or cell nucleus or tissue slice on a slide.
Application:
RNA analysis
Gene mapping
FISH:FLORSCENT IN SITU HYBRIDIZATION
Polymerase chain reaction
Introduction
Cycles
Essential reagent
Process
application
Described in 1985 by Kary Mullis
Nobel Prize in 1993 A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid by means of two flanking oligonucleotide primers used in repeated cycles -generates sufficient for subsequent analysis and/or manipulation
Repeated cycles
denaturation 93°C - 95°C 30sec~1min
annealing 37 °C - 65°C 30sec~1min
extension 72°C 30sec~Xmin94°C 4min
94°C 40sec
55°C 40sec
72°C 50sec
72°C 10min
30~35 cycles
Essential Reagents for PCREssential Reagents for PCR
Template
Primers (18 - 30 nt usual)
dNTPs (dATP, dCTP, dGTP, dTTP)
DNA polymerase (heat stable, e.g., Taq)
Suitable buffer(Mg2+ ,K +, pH)
DNA/RNA from cells, blood, hair root, saliva, semen…..
Forward and reverse primer
Single-stranded oligonucleotide
APPLICATIONS OF PCR
Cloning of genes or gene fragments Genetic diagnosis - Mutation detection Paternity testing Mutagenesis to investigate protein function Quantitate differences in gene expression Identify changes in expression of unknown
genes
Recombinant DNA technology
Definition
Recombinant DNA Tool KitRecombinant DNA Tool Kit
Process
Applications
genomic library
cDNA library
Cell-based DNA amplification
5’ ...GAATTC... 3’ ...CTTAAG...
...GAATTC... 3’
...CTTAAG... 5’
Eco RI Eco RI
Separation of restriction fragments
(P)
-OH5’ G3’ CTTAA
OH
(P)
AATTC 3’G 5’
(P)
-OH
5’ G3’ CTTAA
OH
(P)
AATTC 3’G 5’
anneal anneal
5’ GAATTC3’ CTTAAG
DNA Ligase
Recombinant DNA
Technology by which a DNA molecule is constructed in vitro from segments from more than one parental DNA molecule.
Recombinant DNA Tool KitRecombinant DNA Tool Kit
Restriction Enzymes Vectors (pieces of DNA that allow
maintenance in a cell) Selectable markers Transformation of cells Other enzymes that can be used in vitro -
Ligase, Polymerase, etc.
Cloning VectorsCloning Vectorsdefinition
types
The DNA molecule into which DNA fragment has been cloned,capable of replicating in a special host and thereby propagating the cloned DNA fragment.
Three essential features:
Origin of replication
Dominant selectable marker
Single restriction sitesPlasmid
Bacteriophage lambda
Cosmid
BAC(bacterial artificial chromosome)
YAC (yeast artificial chromosome)
StepsSteps
Prepare a vector. Prepare insert DNA. Connect the two into
recombinant DNA. Transfer recombinant DNA
into host cell (bacteria) Select for positive bacteria
which took up the vector containing the insert DNA.
Propagate the positive bacteria
DNA LibraryDNA Library Libraries are constructed by inserting a variety of
sequences into a cloning vector, and simultaneously maintaining all of these clones; each clone representing an individual insert.
Genomic Library: randomly cut the genome into appropriate size for vector, mix and ligate with vector, replicate in host. Each clone (a vector with an insert) represents a unique region of the genome, but each region of the genome is represented equally in overlapping clones.
cDNA Library: isolate mRNA from a cell or tissue type, convert into DNA, and ligate with vector. Each clone represents a DNA copy of an mRNA, and each mRNA is represented in the library based on its level of transcription.
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